Arabidopsis 2010: An Isotope-Assisted Quantitative Phosphoproteomics Approach to AtHK1-Mediated Osmosignaling in Arabidopsis thaliana

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INTELLECTUAL MERIT As sessile organisms plants have evolved multiple mechanisms to sense and respond to many different environmental inputs, including abiotic conditions (eg. light, water availability) and biotic factors (eg. insects, pathogens). This project will advance our understanding of plant response mechanisms by defining the specific molecular steps involved in transmitting environmental signals to plant cells. Using the model plant Arabidopsis thaliana and advanced techniques in mass spectrometry (isotope-assisted quantitative phosphoproteomics) this project will identify proteins that are modified by phosphorylation as part of important signaling pathways. The initial emphasis will be on AtHK1, a plasma membrane histidine kinase that acts as a receptor for sensing and responding to changes in water availability (drought) to plant cells. This protein is one of a family of several dozen histidine kinases that initiate short- and long-term responses to changes in plant hormones and environmental parameters. Histidine kinases act, at least in part, by a series of phosphotransfers between histidine and aspartyl residues within three different proteins. These modifications (phosphorylations) ultimately result in large-scale changes in transcription, metabolism, cell division and differentiation. In addition to histidine and aspartate phosphorylation, genetic studies of drought responses have implicated a calcium-dependent signaling cascade that includes a serine/threonine protein kinase. No clear model has emerged for integrating these chemically distinct protein phosphorylation systems in the overall response to water availability. In this project, quantitative phosphoproteome measurements via in planta metabolic labeling with non-radioactive heavy isotopes and tandem mass spectrometry will be used, together with mutants displaying various drought-related phenotypes, to comprehensively delineate components of the drought signaling pathway in Arabidopsis thaliana. Previously supported research has developed and refined robust quantitative proteomics technologies using isotope-assisted quantitation by mass spectrometry. These advanced methods will enable identification of groups of phosphoproteins that act in concert in response to important environmental changes, such as drought. The quantitative proteomic measurements will simultaneously provide insights into the response specific for water sensing as well as define a useful paradigm for applying this technology by members of the plant community. BROADER IMPACTS It is well recognized that drought represents the most severe limitation to providing an adequate supply of food for the world population. This project will identify key protein modifications that plants use to sense and respond to changes in water availability, and further develop and make available sophisticated mass spectrometry technology. In order to educate the public and our future leaders on why such expensive equipment and technology are required for research, the PI, Director of the UW Biotechnology Center, and an outreach staff have an established record of providing educational opportunities and activities for the community at large. In the current grant period, a new program will be developed aimed at the general area of "Measuring Molecules". The goal is to demystify mass spectrometers and demonstrate how they provide amazing sensitivity and power for detecting small amounts of good and bad compounds in our environment. The program will start with a discussion of the familiar uses for these instruments, such as at airports to screen for explosives, and on the planet Mars, for reporting on extraterrestrial molecules. The intention is to also develop and use classroom exercises at the high school and undergraduate level, for hands on experience with a new generation of mass spectrometer microscopes, including a MALDI-TOF/TOF that was obtained previously with an NSF MRI grant. Ultimately, through these activities there is hope to instill in the public a better appreciation for the theory and application of advanced technologies that are becoming ingrained in daily life.